Disinfectant dental cements containing liposomes

Abstract

The present invention relates to the field of dental cements used for filling, more particularly to dental cements comprising liposomes giving them a disinfectant power.

Description

The present invention relates to the field of dental cements used for pulp filling, more particularly to dental cements comprising liposomes giving them a disinfectant power. State of the art When a pulp canal infection is diagnosed, the practitioner must disinfect and then fill the canals. The treatment protocol is carried out in several stages. First, the canals are disinfected and shaped, then endodontic cement is used to fill them. After about a week, the canal is unsealed to half its length to allow for post placement, either immediately or 7 days later. The canals are therefore partially exposed to the external environment again during unsealing and post placement, with a risk of bacterial inoculation for approximately 15 days after the start of treatment. Dental cements are used at the end of endodontic treatment (or "root canal therapy"). These cements aim to seal a gutta-percha cone or to plug the pulp canals to ensure the stability of the tooth. However, as mentioned previously, bacteria (residual or inoculated) frequently develop under the cement, leading to infections that result in endodontic treatment failure. The preferred solution is then lengthy and expensive endodontic retreatment with a low success rate, or tooth extraction leading to an implant, which is mutilating, complex, and costly. To prevent post-filling infections and their consequences, various solutions have been proposed, such as UV irradiation or the addition of antiseptic compounds in cements

[12] . As an example, application WO2006 / 070376 describes an endodontic cement comprising nanoparticles made of cationic polymers associated with quaternary ammonium compounds. This cement eliminates Streptococcus bacteria in an in vitro contact test. A similar approach is described in application WO2015 / 004450, but for a bone cement, and the authors propose adding antibiotic-loaded liposomes. None of the solutions proposed in the prior art are effective against all types of bacteria responsible for dental infections, particularly when they are present in biofilm form

[13] . A 2004 study demonstrated the benefit of combining chlorhexidine with a trioxide aggregate endodontic cement Mineral (or MTA for "mineral trioxide aggregate") with a pH of 11.5 to improve the disinfecting power of cement. However, while the presence of chlorhexidine improves the disinfection of E. faecalis bacteria, the effect on biofilms remains limited [12,13]. Current endodontic cements on the market generally have, at best, poor or nonexistent antiseptic properties. With bone cements combined with liposomes, the disinfection problem is very different because (i) the stresses (sealing cement) are very different from those associated with a dental cement (filling cement), (ii) the type of bacteria to combat is different, and (iii) the nature of the cement is different (PMMA-type bone cement versus mineral dental cement). 11. There is therefore a real need

[14] for a dental cement with disinfectant properties, effective against bacteria responsible for dental infections, particularly when they are in the form of a biofilm. Such a cement must, in particular, ensure complete and lasting disinfection against bacteria in biofilm form, and must not be toxic. Advantages of the invention The present invention proposes a new type of endodontic cement which allows for long-term disinfection of the root canal system, the periapex and the dentinal tubules, thanks to the association of liposomes with disinfecting power. DETAILED DESCRIPTION OF THE INVENTION The inventors have developed endodontic cement compositions containing liposomes and have demonstrated their ability to release antiseptics for at least 15 days, the time that usually elapses between disinfection and the final filling of the pulp canals, and up to a month, if necessary depending on the therapeutic protocol implemented. A first object of the invention relates to a dental cement comprising liposomes and having a disinfectant power against bacteria responsible for dental infections, said liposomes being either liposomes loaded with bactericidal agent or other active molecule, or uncharged cationic liposomes. The dental cements according to the invention have a disinfectant power against the bacteria responsible for dental infections, that is to say sufficient to eliminate them, and in particular those which resist the high pH (pH=12) induced by cements based on calcium hydroxide, and which are responsible for post-filling infections. For the purposes of this invention, "disinfectant power" means the ability of a substance to inhibit or kill undesirable microorganisms by altering their structure or metabolism, regardless of their physiological state, in order to reduce their number. 11 There are several methods for measuring the disinfecting power of an endodontic cement, including the direct contact test method and the diffusion method in agar medium (diffusion test in agar, or agar diffusion test). For the direct contact test method, disinfectant power is assessed by measuring the optical density of a solution containing bacteria (for example, at 650 nm for E. faecalis). The lower the optical density, the fewer bacteria are in the solution and the more effective the substance is as a disinfectant. In the case of the agar diffusion method, the disinfectant power is evaluated by measuring the inhibition zone (in mm). The larger the inhibition zone, the higher the disinfectant power. Thus, in a preferred embodiment of the invention, dental cements have a disinfectant power evaluated as follows: either by measuring the bacterial concentration at 7 days using the direct contact test method, this bacterial concentration being less than 10⁵ CFU / mL, preferably less than 10⁴ CFU / mL, and most preferably less than 1000 CFU / mL. This test can be performed on planktonic bacteria or on bacterial biofilm. either by measuring an inhibition zone using the diffusion method in agar medium 24 h to 48 h after inoculation of bacteria and placement of endodontic cements, the inhibition zone being greater than 9 mm, preferably greater than 12 mm. The bacteria responsible for dental infections, particularly those found in the pulp canals, include Streptococcus, Actinomyces, Enterococcus and Propionibacterium strains, and especially Enterococcus faecalis, which is resistant to pH levels above 11.5. Up to 12 different species can be found in these canals. For the purposes of this invention, "dental cement" means any type of cement suitable for temporarily or permanently sealing a dental canal or pulp chamber. The most commonly used dental cements are calcium hydroxide or calcium silicate based, but any cement based on calcium disilicate or trisilicate, hydroxyapatite, silicone, zinc oxide-eugenol, resin, alginate, and / or collagen may also be used. In particular, commercial cements lacking disinfectant properties may be used to prepare the cements according to the invention. Commercial cements include, for example, commercial cements Sealapex™ and Apexit™, BIOROOT™ RCS™, MTA™, Pulp canal sealer™, AH 26™, IROOT™ SP, TOTAL FILL™... A particularly simple temporary cement can consist solely of calcium hydroxide and water or prepared using these two components. In the context of the present invention, cement is used as a matrix (or support) into which liposomes are added. In a preferred embodiment, the cement used is based on calcium hydroxide or on minerals belonging to bioceramics. The liposomes associated with the dental cement can be of different types. Their properties, alone or in combination with active compounds such as bactericidal agents or any other active molecule, impart a disinfectant power to the cement according to the invention. Particularly suitable liposomes are composed of cationic, neutral, or anionic lipids comprising charged chemical entities selected from: ALN-TEG-Chol: 8-(cholest-5-en-3p-xyloxy)-3,6-dioxaoctanyl alendronate, CHEMS: Cholesterol hemisuccinate, Chol: Cholesterol Con-A: Concanavalin A, DC-Chol: 3-p[N(NlNl-dimethylaminoethane-carbamoyl] cholestérol, DCP: Dicetyl phosphate, DDAB: Dimethyldioctadecylammoniumbromide, DMPC: l,2-dimyristoyl-sn-glycero-3-phosphocholine, DMPG: 1,2-dimyristoyl-sn glycero-3-phospho-(l'-rac-glycerol), DOPC: l,2-dioleoyl-sn-glycero-3-phosphocholine, DOPE: l,2-dioleoyl-sn-glycero-3-phosphoethanolamine, DODAB (bromure de diméthyldioctadécylammonium): DOTAP: l,2-dioleoyl-3-trimethylammonium-propane, DPPA: l,2-Dipalmitoyl-sn-glycero-3-phosphate, DPPC: 1,2-dipalmitoyl-snglycero- 3-phosphocholine, DPPG: l,2-dipalmitoyl-sn-glycero-3-phospho-(l'-rac-glycerol), DPTAP: Dipalmitoyl trimethylammoniumpropane, DSPC: l,2-distearoyl-sn-glycero-3-phosphocholine, DSPE: l,2-distearoyl-sn-glycero-3- phosphoethanolamine, PC: Phosphatidylcholine, PEG: Poly(ethyleneglycol), PI: Phosphatidylinositol, PS: Phytosphingosine, SA: Stearylamine, EPC : l,2-dioleoyl-sn-glycero-3-ethylphosphocholine DODAc, 1,2-dimyristoyloxypropyl-3-dimethyi-hydroxyethyl ammonium DMRIE, 2,3-dioleoyloxy-N-(2(sperminecarboxamide)ethyl)-N,N- dimethyi-1 propananninium DOSPA.dioctadecylamidoglycylspermine DOGS, 1,2-dimethyl-dioctadecylammoniumbromide DDAB, 2-dioleyl-3-N,N,N- trimethylaminopropanechloride DOTMA, 1,2-dimyristoy!-3- trimethylammoniumpropane DMTAP, 1,2-distearoyi-3- trimethylammoniumpropane DSTAP, 1,2-Dioleoyl-3-dimethylammonium- propane phospatidylchoiine, phosphatidylethanolamine, sphingomyelin, phosphatidic acid, phospatidylglycerol, phospatidylserine phospatidyiinositol DOBAQ : N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propan-l -aminium Phospholipon 90G phytosphingosine In a first embodiment, the liposomes are loaded with a bactericidal agent or other active molecule. These liposomes can be cationic, neutral, or anionic. They are loaded directly or indirectly with an antiseptic. The liposomes can be loaded after their formation (active loading) or during their formation (passive loading). The term "bactericidal agent" refers to at least one bactericidal agent, but it may also refer to a combination of several bactericidal agents. The bactericidal agent may be chosen from among all topical antiseptics and / or antibiotics that have demonstrated bactericidal activity. In particular, it may be chosen from the compounds listed in ATC class D08 of the classification established by the WTO, which corresponds to "antiseptics and disinfectants." Examples of antiseptics suitable for dental use include copper oxide, zinc oxide, silver, chlorhexidine, triclosan, PVP iodine, QPE1, chitosan, nisin, NaOCl, cationic polymers, fluoride, and bromide. For the purposes of this invention, "other active molecules" means any molecule having a bactericidal, bacteriostatic or other activity complementary to the bactericidal agent itself. The encapsulation of antiseptics or other active molecules in liposomes allows the latter to be protected against inhibition caused by biological fluids, but also allows for controlled and effective release over time. Liposomes allow for better penetration of the bacterial biofilm [10;11] and therefore greater antibacterial efficacy. Indeed, they can fuse with the biofilm walls and thus deliver active molecules to the heart of the biofilm, in addition to mechanically destabilizing bacterial membranes. In a preferred embodiment, the liposomes are cationic liposomes and the active molecule is an antiseptic such as chlorhexidine. In another preferred embodiment, the liposomes are cationic liposomes and the active molecule is an antibiotic such as a penicillin family antibiotic, for example amoxicillin. In another preferred embodiment, the liposomes are cationic liposomes and the active molecules are an antibiotic / antiseptic combination, for example nisin / tetracycline. In another preferred embodiment, liposomes are an association of liposomes of different natures (cationic, neutral, anionic) and the active molecules are an antibiotic and / or an antiseptic. In a second embodiment, the liposomes are cationic and uncharged. When cationic liposomes are not loaded with active molecules, their disinfectant power relies on their ability to fuse with biofilms. By fusing with the biofilm walls and bacterial membranes, they disrupt both the biofilm and the bacterial membranes, leading to bacterial destruction. Regardless of the specific formulation, the addition of liposomes to dental cements is particularly advantageous for bacterial disinfection because liposomes have an affinity for bacteria and can diffuse into the pulp canal system, dentin tubules, and periapex. Liposomes allow for a more controlled diffusion of the antiseptic or bactericidal agent than if the agent were present alone in the cement. In all cases, whether the liposomes are loaded or not, their size must be small enough to allow them to penetrate the channels and tubules. The size Tubular wavelengths generally vary between 100 nm and 3200 nm, with an average of 800-1200 nm. Thus, the size of the liposomes used in the present invention can vary between 20 and 8000 nm. In a particular embodiment, their size is less than 6000 nm, preferably between 600 nm and 50 nm, or even between 400 nm and 50 nm, more preferably less than 300 nm and quite possibly between 100 nm and 200 nm, for example 150 nm. The properties of disinfectant cements vary depending on the chosen combination: the type of cement, the type and concentration of liposomes, and the type and quantity of active molecule. In this respect, a disinfectant cement for dental use will differ from one for bone restorations. Furthermore, the type of cement, as well as the type and size of the liposomes, can influence liposome diffusion and therefore the release of active molecules from the cement upon contact with the tooth. It is important to control these parameters to manage the final quantity of antiseptics or other active molecules released. In a preferred embodiment of the invention, the cement is based on calcium hydroxide or bioceramics, and the liposomes are of the type "DOPE liposome", "EPC liposome" or "DODAP liposome", as defined in the experimental part (Example 4. Section 2.). In another preferred embodiment, the dental cement comprises a Pluronic-type sequenced copolymer as a surfactant. Surfactants promote the homogeneous distribution of liposomes within the cement. The following cements are particularly preferred: Sealapex® type calcium hydroxide-based cement with DOPE-type liposomes Apexit® type calcium hydroxide-based cement with EPC-type liposomes Total fill® type calcium silicate cement with DODAP type liposomes A second object of the invention relates to a method for preparing a dental cement as defined above, comprising: a) the preparation of liposomes with a size between 8000 nm and 20 nm, b) the mixing of said liposomes with a dental cement in a proportion between 0.5 and 30%, preferably between 1% and 5% liposomes (w / w) In a preferred embodiment, a surfactant is added to the liposome preparation before mixing with the cement. This prevents miscellification and subsequently allows the hydrophilic cement base to mix with the hydrophobic liposomes. Among the surfactants suitable for this use, we can mention the pluronics L31, L61, F68, F127, L43, L44, L62, L64, P85, P84, P104, P123. The addition of surfactant promotes the homogeneous distribution of liposomes in the cement and allows good reproducibility of product quality, and in particular to achieve the quality required for the specification of products for dental use. When liposomes are loaded with active molecules (antiseptics or other), the process further includes a step of including (or loading) these molecules into the liposomes before mixing the liposomes with the cement. The percentage of liposomes included in the cement can range from 0.5% to 30% by weight, particularly below 20%, typically between 1% and 10%, and preferably between 1% and 5% by weight. Ideally, this percentage should be between 2% and 4%, but may vary depending on the size of the liposomes and their antiseptic content. In all cases, the disinfectant power of the cement must be controlled. A third object of the invention relates to the use of a dental cement as defined above for dental filling, in particular of the root canal, periapex and / or dentinal tubules. Endodontic cements, as previously defined, can be used for temporary or permanent fillings depending on the treatment protocol followed by the dentist. Thanks to their disinfectant properties, they help prevent deep infections of the root canals, which are difficult to treat and require further intervention. A fourth object of the invention relates to a method for evaluating the disinfectant power of a dental cement, consisting of: (i) arrange, in a two-compartment container, a) a solution comprising bacterial strains likely to be responsible for a dental infection in the lower compartment b) a layer of dental cement whose disinfectant power we wish to test in the upper compartment, the two compartments being separated by a membrane perforated with pores whose diameter is between 8000 nm and 50 nm; ii) incubate the container at 37°C for a chosen duration; iii) assess the presence of bacteria in the lower compartment. This experiment can be performed by preparing two-compartment containers separated by a membrane with pores of an appropriate diameter. Alternatively, commercial devices such as the Transwell® plates sold by Corning® can be used. The diameter of the membrane pores is custom-chosen. To reproduce the diameter of the tubules, it will be chosen between 50 and 8000 nm, preferably between 300 and 800 nm and more specifically between 50 nm and 400 nm. The incubation time will generally be between 2 and 48 hours. A person skilled in the art will know how to adjust this time according to standard microbiology protocols. The assessment of the presence of bacteria will be carried out by one of the microbiological techniques known to a person skilled in the art, for example by measuring the optical density of the solution by spectrometry. This test is particularly relevant for evaluating the disinfecting power of a dental cement, since the nanopores in the membrane, due to their size, mimic dental tubules. If bacteria are eliminated, this suggests that the cement will be effective in disinfecting the tubules. Finally, this test can be used to measure the disinfecting power of endodontic cement either against planktonic bacteria or against a bacterial biofilm, depending on the bacteria that will be added to the solution placed in the lower compartment. The present invention will be better understood by reading the following examples, which are provided by way of illustration and should in no way be considered as limiting the scope of the present invention. DESCRIPTION OF THE FIGURES Figure 1: Diagram of a container adapted for the method of evaluating the disinfectant power of a dental cement. Figure 2: Representation of the size distribution of the liposomes formed. Figure 3: Representation of the release kinetics of cements containing different types of rhodamine-loaded liposomes. (A) Sealapex® type calcium hydroxide-based cement (B) Apexit® type calcium hydroxide-based cement. The types of Liposomes used are illustrated as follows: DOPE: square; EPC: triangle; DODAP: diamond; control without liposome: circle. Figure 4: Representation of the percentage of rhodamine release contained in cements according to the type of liposomes at 15 days. Ctl = control without liposomes. EXPERIMENTAL SECTION Example 1: Preparation of cements containing liposomes 1 - Preparation of liposomes Liposomes were formed from the different lipids of interest described above using different classical techniques: sonication or hydration of a lipid film [9]. In all cases, residual organic solvents were removed to recover a pure powder, which was then resuspended in a biologically compatible solvent (usually water) to generate liposomes. This was achieved using evaporation techniques, which are unnecessary if the liposomal detergent is delivered to the aqueous phase at a satisfactory minimum concentration. For this proof of concept, medium- to high-quality reagents were required (>97.0% purity). To prepare cements for dental use, this will need to be improved to a minimum >99.0% purity (purification by HPLC). One of the protocols used follows, with an adaptation, the previously published protocols [1, 2, 9]. In the preparation of the liposomes, a weight-to-weight ratio of the desired lipid combination was achieved by precisely weighing the given detergent lipid before mixing it in a large round-bottom flask. Five mL of organic solvent (chloroform, HPLC grade) were added, and the suspension was vortexed until the lipids were completely dissolved. The same flask was attached to a rotary evaporator kit equipped with a vacuum pump rotating at one revolution per second. The system was maintained at a constant temperature, typically 60 °C, using a water bath; this temperature was determined by the phase transition of the lipid mixture of interest. Upon removal of all organic solvents, sterile MQ deionized water was added to give a concentration of 5 mg.mL-1. The suspension was then maintained for a further 30 minutes to allow liposomes to form. Next, the suspension was scooped and extruded ten times under 8 bar nitrogen pressure through a Lipex-type extruder. The mixture was then extruded a second time ten times through 400 nm polycarbonate membranes and a third time ten times through 100 nm polycarbonate membranes to ensure greater homogeneity in the liposome size range. Finally, the suspension was centrifuged at 100,000 g for one hour (at 4°C) to pellet the liposomes and remove excess water in preparation for loading. In these tests, liposomes were prepared from DOPE, EPC, DODAP and PEG. 2- Inclusion of bactericidal compounds The bactericidal compound is loaded into liposomes via active or passive pathways, depending on the chemical properties of the detergent lipid and the chemical compound in question. Previously published studies have examined liposomal loading with the antibiotic gentamicin sulfate to form a bone cement, finding an improved therapeutic index of the antibiotic, as well as greater accumulation at sites of interest [2]. A related study examined the loading of the antifungal amphotericin B into liposomes to generate a bone cement for active treatment [3]. Particularly interesting antiseptics include copper oxide, chlorhexidine, triclosan, PVP iodine, and nisin, each of which can be charged according to previously published protocols. Those charged by active means (generally those of a hydrophilic nature) were added using an externally generated gradient, particularly by modifying the pH [4] or electrochemically [5, 6]. Passive charging of chemical compounds is a simpler process, and here, the chosen chemical compounds were dissolved in the solvent / organic phase if hydrophobic, or in the aqueous phase if hydrophilic. In all cases, the concentration of the liposomal samples was considered at every stage to help maintain the mixture at levels that account for the CMC (critical miscellation concentration). In lipid studies, the CMC is the concentration above which the solution will begin to generate surfactant micelles. Generally, the CMC is determined by the hydrophobic-hydrophilic disintegration in the sample; therefore, the greater the disintegration, the lower the CMC. The chemical basis of the lipid molecule also affects the CMC through its segment lengths, whereby longer segments result in a lower CMC. All these factors were taken into consideration for the present study. 3 - Determination of the size and homogeneity of liposomes Laser diffraction was used to determine the average diameter of the charged liposomes and to confirm that the liposomes were not damaged and that their size was on the order of 100 to 150 nm. This analysis, shown in Figure 2, was performed using a Beckman Coulter N4 Plus particle size analyzer, under conditions corresponding to those published previously [2]. Once the liposomes were loaded, a surfactant (2% wt / w) depending on the type of liposomes chosen was added. Various types of surfactant were used, including: Pluronics L31, L61, F68, F127, L43, L44, L62, L64, P85, P84, P104, and P123. 4 - Mixing the liposomes with the cement and analyzing the resulting product Once the liposomal nanoparticles were formed, they were incorporated into the chosen cement, mixed, and prepared for testing. These techniques follow, with the indicated adaptation, previously published work [2]. In these tests, the cements used were the commercial Sealapex® and Apexit® cements. After the preparation of the liposomes, the final cement is obtained by mixing the liposomal solution with the cement to obtain a final liposome concentration of 3% (w / w). The final cement can be obtained alternatively by incorporating lyophilized liposomes into the endodontic cement powder. Transmission electron microscopy was used to visualize the liposome / cement mixture to better understand liposomal dispersion within the matrix. The technique can be performed as follows: a 1:1 ratio of liposome nanoparticles was prepared with 4% w / v aqueous uranyl acetate, and the mixture was incubated for 60 minutes. The liposomes were then concentrated by centrifugation as described above and resuspended in 10 mL of a given cement, or the same volume of water as the control. The suspension was further diluted to a final concentration of 10% v / v and 10 / z L, and added to a Formvar carbon film supported on a 400-mesh nickel grid (EM Systems Support Ltd). The support was air-dried, and each undiluted cement was also inspected. The dried nickel grids were analyzed by 80 kv transmission electron microscopy (Philips CM12 TEM), and the images were saved for analysis. Example 2: Study of the release of charged molecules in liposomes The experiments described here concern the detection of the release of molecules encapsulated in liposomes and the evaluation of the antiseptic properties of the endodontic cement formed. The tests were carried out with cement mixtures. containing in the final volume each of 0%, 1% and 3% (wt / wt) of loaded liposomes. 1 - Release of nanoparticles a. Simple release The endodontic cements used in these experiments are commercially available. Specifically, they are Sealapex® and Apexit® cements. They were prepared according to the manufacturer's instructions. For each endodontic cement, the final volume of each liposomal nanoparticle formulation was 0%, 1%, and 3%. The cement was applied to the bottom of the well in a 96-well tray in triplicate. After complete setting of the cement, 1 mL of PBS pH 7.2 was added to each well, then withdrawn and replaced with fresh solution according to the following schedule: 4 h, 8 h, 12 h, 24 h, 36 h, 48 h, and then once every 24 h for 15 days. A preliminary release study was conducted using cements containing rhodamine-loaded liposomes, without the addition of surfactant. The results of this study are presented in Table 1 below: they show the release of rhodamine in relatively small quantities. PF G Biodentine Apexit 72h .0875484 0.134 2 0.04554 0.02861 Final shad relayRuoc in s. of the starting dose 0.1750968 0.2684 5.36800% 0.09108 0.05722 epc 7?" 0.1291 0.2582 Apexit 0.1086 0.2172 4.34400% DOPE 72h final release released! as a % of the starting dose 0.0214 Biodentine 0.08266 0.16532 3.30640% sealapex 0.1347 0.2694 5.38800% Apexit 0.107012 0.214024 These results led the inventors to improve cement preparation by adding a surfactant to the liposomal solution before mixing with the cement. The cements used in the remainder of the study contain a surfactant. b. Release through a porous membrane The cement was placed in the upper compartment of the Transwell® insert on the porous membrane (400 nmj). After the cement set time, the lower compartment was filled with 3 different fluids: water, physiological fluid or artificial dental fluid (artificial cerebrospinal fluid). The presence of liposomes was measured by spectrophotometry at 1 h, 4 h, 24 h, 48 h, 72 h, 2 weeks and 3 weeks. The same experiment was performed with E. faecalis strains placed in the lower compartment (see Transwell® insert test). Three E. faecalis strains were used: one from ATCC 29212 and two others, RW35 and RN44 (see below for the culture and identification method). Example 3: Evaluation of the antibacterial properties of cements containing liposomes 1 - Direct contact test The antibacterial efficacy of nanoparticles was tested against E. faecalis, a common and resistant intraductal pathogen. To support the antibacterial effect of the nanoparticles, they were tested after immobilization. To define the mode of action, the effect was further evaluated in bacterial suspension. a. Preparation of the bacterial suspension E. faecalis was cultured overnight in 5 mL of brain-heart perfusion broth (BH1, Difco, Detroit, MI, USA) at 37°C under aerobic conditions. The top 4 mL were transferred to a new test tube and centrifuged for 10 min at 4,165 x g. The supernatant was discarded, and the bacteria were resuspended in 5 mL of PBS and shaken for 10 s. The optical density of the bacterial suspension was adjusted to 650 nm, corresponding to a transmittance of 90 T (0.5 on the McFarland scale: 1.5 × 10⁸ CFU / mL). b. Antibacterial effect of immobilized liposomes The surface antimicrobial effect of endodontic cement incorporating 0%, 1%, or 3% (w / w) liposomes was then evaluated. A microtiter plate (plate with flat bottom of 96 wells, Nunclon, Copenhagen, Denmark) was positioned vertically and the side walls of 8 wells were coated with similar quantities of the material (lOmg) tested according to the procedure described above. Ten µL of the bacterial suspension were added to the surface of each cement and to empty wells. After inoculation (at 37°C at moisture saturation) for 2, 5, 20, and 60 minutes, 240 µL of PBS were added to each well. After stirring for 1 minute using a pipette, the resulting solution was transferred and serially diluted. Bacterial survival was measured by culturing 20 µL aliquots of the transferred solutions onto Triptic soy agar plates after serial dilution by a factor of 10. After incubation for 24 h at 37°C, the colonies on the plate were counted, and the CFU / mL was calculated. The presence of bacteria was measured by spectrophotometry (at 1 h, 4 h, 24 h, 48 h, 72 h, 2 weeks and 3 weeks). 2. Test in Transwell® insert A membrane with 400 nm pores was used to evaluate the diffusion of liposomes through these nanopores; these nanopores, due to their size, simulate dentine tubules. In a 96-well plate, the upper compartment was filled with 10 mg of cement including or not containing liposomes, and the lower compartment was filled with a solution including the bacterial strains. Three strains of E. Faecalis were tested, namely strains ATCC 29212, RW35 and RN44. The evaluation of disinfectant power was measured in the same way as in the direct contact test. 3. Diffusion test in agar A further test was carried out to demonstrate the disinfecting power of our endodontic cement containing liposomes and to compare it to that of cements without liposomes (which have limited disinfecting power, particularly on dental biofilm). The test was performed in a petri dish filled with a 10 mL layer of sterilized Muller Hinton agar. Wells 4 mm in diameter were formed in this agar layer to hold endodontic cements prepared under the same conditions as above. In this experiment, antibacterial activity was evaluated against strains of Pseudomonas aeruginosa, Enteroccus faecalis, Staphylococcus aureus and Escherichia coli. The bacterial strains were cultured at 37°C for 24 h in MH agar to produce a turbidity of 0.5 on the McFarland scale, corresponding to a concentration of 10.8 CFU / mL. A layer containing these bacteria was deposited to cover the freshly mixed cements. The plates were kept at room temperature for 2 h and incubated at 37°C for 24 h. Monitoring was performed at 4 h, 8 h, 12 h, 24 h, 36 h, 48 h, and then once every 24 h for 15 days: the zone of inhibition in mm was measured with calipers (visual test). 4. Modified direct contact test (biofilm) This test demonstrates the disinfecting power of an endodontic cement against a bacterial biofilm (the predominant form of bacteria in the pulp canal). The biofilm was formed on 4 mm x 4 mm x 1.5 mm (width x length x thickness) slices of bovine incisors using a low-speed diamond disc under irrigation. The samples are soaked in 17% EDTA for 3 minutes to remove dentin sludge and then sterilized at 121°C for 20 min. A standard strain of E. faecalis (ATCC 51299) was used for bacterial biofilm formation. E. faecalis was cultured under the same conditions as before. Bovine tooth slices were placed in wells of a 24-well plate. 220 µL of inoculum was added to each well, followed by 1.8 mL of sterile BH1 solution. The wells were cultured at 37°C with 5% CO2 for 14 days. The supernatant was completely replaced every 48 hours with fresh sterile BH1 solution. The endodontic cements prepared under the conditions mentioned above are placed on the sections of bovine tooth covered with biofilm. The endodontic tooth / cement samples are placed in a new 24-well plate, and stored at 37°C with 5% CO2 for 2, 7 and 14 days. After these incubation periods, the remaining biofilms are transferred to tubes containing µL of saline solution. The tubes are shaken with an ultrasonic sonicator for 30 seconds at 40 W to detach the biofilms. The measurement of the number of bacteria (CFU / mL) is carried out as previously described. Example 4: Evaluation of the percentage release of molecules contained in liposomes mixed with cement 1 - Objective of the experiment The following experiment aims to evaluate the percentage of release of a fluorescent molecule (rhodamine) incorporated into liposomes mixed with endodontic cement. In practice, it consisted of comparing i) the release obtained from 10 mg of cement containing 3% liposomes (by weight) containing rhodamine (which corresponds to the addition of 1% lipids by weight to the cement) versus ii) the release obtained with a cement not comprising a liposome but only rhodamine (fluorescence alone). The base cements used in this experiment were the commercial cements Apexit®, Sealapex®, and Bioroot® RCS. The liposomes were prepared from the following lipids: EPC, DOPE, and DODAP. 2 - Preparation of liposomes The liposomes were prepared according to the method described above. The experiment was carried out with the following compositions: "DOPE liposome" (DOPE, HSPC), "DODAP liposome" (DODAP, PC, Cholesterol), "EPC liposomes" (EPC, HSPC) in the following respective proportions: (5:2:3 molar ratio), (3:7 weight ratio), (35:65 weight ratio). A rhodamine solution (0.04 nMol) is used to hydrate the lipid film and obtain liposomes containing fluorescent molecules (liposome loading). 3 - Preparation of the cement comprising rhodamine-loaded liposomes After obtaining the liposomes, they are mixed with Pluronic L31 surfactant at 2% w / w. The previously prepared fluorescent liposome solution was then incorporated into the various cements tested. 100 mg of a given cement were mixed with 1 mg of lipids. 10 mg of the lipid / cement mixture was then arranged in triplicate in wells of a 96-well plate. To prepare the "liposome-free" control cement, the rhodamine solution was incorporated directly into the cements in the same proportions as those used for the liposome-containing cements (the same amount of rhodamine in the cement). The resulting mixtures were placed in a 37°C incubator with saturated humidity. 4 - Analytical methods used Kinetics of the release study: After complete setting of the material (approximately 4 hours), ImL of PBS (pH 7.2) was added to each well, then removed and replaced with a fresh supernatant solution according to the following kinetics: 4h, 8h, 12h, 24h, 36h, 48h then once every 24 h for 15 days. Liposome size: The size of the liposomes was measured according to the method described above. We also measured the size of the liposomes after release from the cement. Fluorescence assay: The rhodamine solution serves as a standard for the fluorescence assay. At the time of the assay, the supernatants were collected from each well and transferred respectively to a well of another plate used for the assay. The assay is performed by spectrophotometry (BIOTEK plate reader). 5 - Results The results of this study are presented in Figures 2 and 3. They were incorporated into different types of cement, either liposomes encapsulating rhodamine, or unencapsulated rhodamine. Rhodamine release: The results show that endodontic cements incorporating liposomes exhibit greater release in both quantity and duration compared to cements without liposomes (curves marked with a circle represent cement without liposomes). This property is particularly important in endodontics, as effective bacterial elimination requires sufficient and sustained release [10; 11]. Thus, these results demonstrate that liposome incorporation allows for the release of up to 19.43% of the rhodamine incorporated into the cement, versus 1.67% when the rhodamine is not encapsulated in liposomes. Furthermore, the entire amount of rhodamine released in liposomes-free cements occurs within 24-48 hours; after this time, the cement releases very little of the fluorescent substance. This innovation therefore allows for diffuse release over a longer period of 15 days. The cement developed achieves a constant minimum inhibition / bactericidal concentration over time, unlike liposomes-free endodontic cements, which reach this concentration for only 24 hours (the release in subsequent days does not allow the minimum inhibition concentration to be reached locally). Finally, the results show that the combination of cement and rhodamine-encapsulating liposomes allows for greater release than the combination of rhodamine and cement, regardless of the type of cement and liposomes used. However, the release profiles differ depending on the lipids and cements used. Finally, we find the same release profiles as in previous studies combining bone cement and liposomes [1]. Furthermore, the formulation can be modulated according to the objective to be achieved, by modifying the nature of the lipids, the nature of the antiseptic and the nature of the endodontic cement. In conclusion, liposomes have been successfully incorporated into endodontic cement to allow for the controlled release (in quantity and duration) of a molecule of interest. This innovation could play a significant role in reducing post-treatment endodontic infections, which affect 44% to 77% of treated teeth. Improved disinfection through this innovation would lead to fewer cardiovascular (heart attack) and respiratory (sinusitis) complications, as well as a reduction in costs associated with endodontic treatment failure (antibiotics, crowns, implants, etc.). Bibliographical references [1] Ayre, N.W., Birchall, J.C., Evans, S.L. and Denyer, S.P. A novel liposomal drug delivery System for PMMA bone cements. J BiomedMater Res Part B. 2015;00B:000-~000. [2] Ayre, NW. Novel approaches to the development of PMMA bone cernent. Thesis, PhD. 2013. Cardiff University. [3] Cunningham, B., McLaren, A.C., Pauken, C. and McLemore, R. Liposomal formulation increases local delivery of amphotericin from bone cernent: a pilot study. Clinical Orthopaedics and Related Research. 2012;470:2671-2676. [4] Mayer, L.D., Bally, M.B. and Cullis P.R. Uptake of adriamycin into large unilamellar vesicles in response to a pH gradient. Biochim Biophys Acta. 1986;857:123-126. [5] Clerc, S. and Barenholz, Y. Loading of amphipathic weak acids into liposomes in response to transmembrane calcium acetate gradients. Biochim Biophys Acta. 1995;1240:257-265. [6] Haran, G., Cohen, R., Bar, L.K. and Barenholz, Y. Transmembrane ammonium sulfate gradients in liposomes produce efficient and stable entrapment of amphipathic weak bases. Biochim Biophys Acta. 1993;1151:201-215. [7] Ayre, N.W., Birchall, J.C., Evans, S.L. and Denyer, S.P. Liposomal Drug Delivery System for Bone Cements fWO 2015004450 Al). United Kingdom, PCT / GB2014 / 052085. 9th July 2014. [8] Beyth, N., Domb, A.J. and Weiss, E.l. An in vitro quantitative antibacterial analysis of amalgam and composite resins. J Dent. 2007;35:201-206. [9] Abolfazl Akbarzadeh, Rogaie Rezaei-Sadabady, [...], and Kazem Nejati-Koshki Liposome: classification, préparation, and applications

[10] Katrien Forier, Koen Raemdonck, Stefaan C. De Smedt, Jo Demeester, Tom Coenye, Kevin Braeckmans Lipid and polymer nanoparticles for drug delivery to bacterial biofilms

[11] Kazuo Yamakami, Hideaki Tsumori, Yutaka Sakurai, Yoshitaka Shimizu, Kohei Nagatoshi & Kenji Sonomoto Sustained inhibition efficacy of liposomeencapsulated nisin on insoluble glucan-biofilm synthesis by Streptococcus mutans

[12] Ted J. Stowe, DDS, Christine M. Sedgley, BDS, MDSc, ​​MDS, FRACDS, PhD, Bryant Stowe, and J. Christopher Fenno, PhD. Volume 30, Issue 6, June 2004, Pages 429-431

[13] NB Faria-Ju nior, M. Tanomaru-Filho, FLCV Berbert & JM Guerreiro-Tanomaru, Antibiofilm activity, pH and solubility of endodontic sealers, Int Endod J. 2013 Aug;46[8]:755-62.

[14] _HARALD M ER1KSEN, L1SE-LOTTE CHURCH Endodontic epidemiology and treatment outcome: general considerations ;

Claims

DEMANDS 1. Dental zicome characterized in that it comprises liposomes and has disinfectant power against bacteria responsible for dental infections, - said liposomes being either liposomes loaded with a bactericidal agent or other active molecule, or uncharged cationic liposomes, - said dental cement being based on calcium hydroxide or calcium silicate, disilicate, calcium trisilicate, hydroxyapatite, silicone or zinc eugenol oxide, and - said cement further comprising a surfactant.

2. Dental cement according to claim 1 wherein the disinfecting power is evaluated by measuring the bacterial concentration at 7 days using the direct contact test method, this bacterial concentration being less than 105CFU / mL, preferably less than 104 CFU / mL, and most preferably less than 1000 CFU / mL, and said bacterial power being evaluated either on planktonic bacteria or on bacterial biofilm.

3. Dental cement according to any one of the preceding claims, wherein the liposomes have a size between 20 nm and 8000 nm.

4. A method for preparing a dental cement as defined in any one of claims 1 to 3, comprising: a) the preparation of liposomes with a size ranging from 20 nm to 8000 nm, b) the mixing of said liposomes with a dental cement in a proportion of between 0.5% and 30% liposomes (weight / weight).

5. A method according to claim 4, wherein the surfactant is added to the liposome preparation before mixing with the cement.

6. A method according to any one of claims 4 or 5, in which the preparation of liposomes include a step of loading said liposomes with antiseptics and / or other active molecules.

7. Dental cement as defined in any one of claims 1 to 3, for its use in the prevention of post-filling infections, in particular of the root canal, periapex and / or dentinal tubules.

8. Method for evaluating the disinfectant power of a dental cement as defined in any one of claims 1 to 3, consisting of: (i) arrange, in a two-compartment container, a) a solution comprising bacterial strains likely to be responsible for a dental infection in the lower compartment, b) a layer of dental cement whose disinfectant properties are to be tested in the upper compartment, 5 the two compartments being separated by a membrane perforated with pores whose diameter is between 50 nm and 8000 nm; ii] incubate the container at 37°C for a chosen duration; iif) assess the presence of bacteria in the lower compartment.

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